Summary: 1. Molecular Aspects and Clinical Characteristics of Silent HBV: The uncommon finding of "silent" hepatitis B virus (HBV), characterized by the presence of HBV DNA in serum or liver tissue in the absence of hepatitis B surface antigen (HBsAg), anti-HBc and anti-HBs in serum, is thought to be caused in part by HBV mutants that produce a very low level of HBsAg, which escape detection by currently available serologic tests. This is an issue of blood transfusion safety. HCC and adjacent liver tissues from forty-three patients whose sera were HBsAg-negative, as well as three positive and four negative controls (a total of 87 DNAs) were further studied for HBV DNA using nested PCR, including 9 from various areas of the USA, 22 from Vancouver, Canada, and 12 from China. HBV DNA was detected by PCR in 5 of the 9 patients (55%)from HBsAg-negative patients from the USA, in 12 of the 22 from Canada (55%), and in 12/12 from Qidong, China (100%). Sequencing of the 241-bp S gene PCR product was performed in the patients from the USA and Canada; each was found to contain the expected S gene sequence with one or more nucleotide variations differing from the consensus sequence and from each other. Genotyping was determined using primers for type-specific sequences in the S gene. Replication of HBV was determined by the presence of replicative intermediates (covalently closed circular [CCC] HBV DNA). In HBsAg-positive patients, CCC DNA was found in 3/5 (60%), while in silent HBV, CCC DNA was found in 1/9(11%); in residual HBV, CCC DNA was found in 1/8(12.5%). Among all HCC patients from the USA and Canada who were HBsAg-negative, HBV DNA could be detected in 55%. 2. Detection of HBV DNA in two Plasma Derivatives: Seventeen vials of 5 separate samples (samples A, B, C, D [4 vials of each], and V [one vial]) from 2 sources of alpha-1-antitrypsin were studied for the presence of HBV DNA using Taqman PCR; three out of 4 samples from 1 source had reportedly been found to contain HIV, and one from the other source was reported to have been HIV negative. Validation experiments on the HBV Taqman method were conducted using a standard curve constructed on a dilution series of the WHO HBV DNA reference standard, as well as multiple positive and negative controls. In order to prevent possible contamination, careful decontamination procedures were conducted. In addition, sample extraction and Taqman reagent preparation were conducted in three different rooms for different experiments, including a room that had never been used for HBV DNA PCR (bldg 29/rm 320). Results were: a. The selected primers and probe for Taqman PCR for HBV DNA worked well. All positive controls and negative controls gave reproducible results in each run. The standard curves had a correlation coefficient >0.98. b. Six normal plasma samples extracted in 29/Rm 320, four normal plasma samples extracted in 29A/Rm 1C/14, and three to four "no template negative controls," all tested negative in all runs. c. Each of 4 retention samples of Sample D (from sets #1-4) was extracted and tested separately; each tested repeatedly positive for HBV DNA. Sample B tested repeatedly positive for HBV DNA from three of four sets. Samples A and C tested positive (at low levels) for HBV DNA in samples from two of the four sets (each run in duplicate; total of four positive reactions). An additional retention sample, sample V, tested positive in duplicates of 2 extractions (total of 4 reactions for sample V). d. Sequencing of the PCR products of samples B and D (from set #1) confirmed that both had HBV DNA, and revealed that two of 46 nucleotides differed from the sequence of the WHO HBV DNA reference standard, which rules out possible contamination from the WHO reference standard, which was the only positive sample used in these studies other than the alpha-1-antitrypsin. 3. Early Detection of Hepatitis B Virus Infection: Comparative Sensitivity of HBV DNA Detection by PCR and HBsAg Detection by a New Generation of Assays. Based on tests of the FDA HBsAg lot-release panel in the present study, newer HBsAg tests have sensitivity of 0.07-0.18 ng/ml, compared to 0.13-0.62 ng/ml for 4 licensed tests evaluated in this study. Tests on the WHO HBV DNA standard showed that the viral load at cut-off was 102-364 IU/ml for newer HBsAg tests, and 363-1069 for licensed HBsAg tests. In 90 samples collected during the exponential phase from 10 seroconversion panels, HBsAg tests detected 31-61% and PCR detected 82-99% of samples. In the pre-exponential phase, no sample was detected by licensed HBsAg tests, while PCR detected HBV at 30-80% of these samples. The newer HBsAg tests detected infections 2 weeks earlier and PCR detected HBV infections about one month earlier than currently available HBsAg tests.